使用分裂的角质按钮的孔膜内皮器官培养模型
Summary
这里提出了猪分裂角膜纽扣的制备和培养的分步方案。由于这种通常培养的器官培养模型显示细胞死亡率在15天内，相当于人类捐赠角膜，它代表了第一个模型，允许长期种植非人类角膜，而不添加有毒的dextran。
Abstract
角膜内皮细胞的实验研究与几个困难有关。供体角膜很少，很少可用于实验性调查，因为通常移植需要角膜。内皮细胞培养物往往不能很好地翻译到体内的情况。由于非人类角膜的生物结构特征，栽培过程中的基质肿胀会导致角膜内皮细胞大量流失，长期难以进行栽培。脱毛剂（如 dextran）用于抵消此响应。然而，它们也导致显著的内皮细胞损失。因此，建立了不需要脱肿剂的体外器官培养模型。当地屠宰场的猪眼被用来准备分裂的角膜纽扣。局部角膜发角膜后，角膜的外层（表皮、弓形层、频闪部分）被移除。这大大减少了角膜内皮细胞损失引起的大规模基质肿胀和Descemet膜折叠在较长的培养期，并改善内皮细胞层的一般保存。随后，完全角膜裂解，然后从剩余的眼球和培养中去除分裂的角膜按钮。使用光显微镜在制备后长达15天的随访时间（即第1天、第8天、第15天）评估内皮细胞密度。使用制备技术可以更好地保存内皮细胞层，通过较少的基质组织肿胀实现，从而导致分裂角膜按钮与人类供体角膜相媲美的缓慢和线性下降率。由于这种标准化的有机体典型栽培研究模式首次允许稳定种植至少两周，它是人类供体角膜的宝贵替代品，用于今后研究各种外部因素，对角膜内皮的影响。
Introduction
角膜移植手术是全世界最常见的移植之一。由于人类供体角膜严重短缺，人类角膜内皮细胞的实验研究难以进行。然而，引进灌溉溶液和眼睛内使用的其他物质，眼科粘弹性装置，以及手术器械和技术（例如，乳化仪器和技术，超声波能量）需要临床使用前，对角膜内皮药的影响进行有效和广泛的调查。
除了人类供体角膜之外，几乎没有其他选择可供研究。动物研究模型是非常有价值的，但与此同时，非常耗费资源，并越来越质疑伦理。体外细胞培养的一个主要缺点是它们对人眼的翻译有限。从细胞培养得到的结果可能与体内条件不协调，因为细胞可能经历内皮性等位体过渡（EMT），导致细胞极性丧失和细胞形状和基因变化引起的成纤维细胞样形态表达式2.
而以前的前体模型报告培育期只有120小时，一种新的制备技术，以建立猪角膜内皮器官培养模型，通过培育新鲜猪角膜至少15天最近推出3 ，4，5，6.如果在种植前从角膜上皮和频闪部分（总计约300 μm）去除角膜，则角质的肿胀在分裂的角膜按钮中减少，从而减少内皮细胞损失，并维持良好的内皮细胞层后长达15天，而非分裂的角膜按钮显示显着的内皮细胞损失，由于不均匀的基质肿胀和德塞梅特的褶皱的形成。眼库通常使用渗透消肿剂，如dextran，以减少在移植前角膜肿胀。然而，这些制剂被证明诱导增加的内皮细胞损失7，8，9。
本文旨在以详细的分步协议来可视化这种标准化的体外研究模型，以便未来的研究者能够使用分割角膜按钮对角膜内皮进行研究。该模型代表一种简单的方法来测试眼睛中使用的物质和技术，如眼粘弹性装置、灌溉溶液和超声波能量，或角膜内皮感兴趣的其他程序。
Protocol
本协议遵循我们机构的道德准则。根据我们机构道德审查委员会的章程，在实验之前无需获得道德批准，因为所有猪角膜都是从当地屠宰场获得的。 1. 器官文化 准备猪眼。 从当地的屠宰场，获得猪眼，在验尸后，但在热处理之前被摘除。将眼睛转移到实验室，并在几个小时内处理它们。在运输过程中，在加工前将眼睛保持在室温（约21°C） 下。 使用眼…
Representative Results
提出的解剖技术意味着部分去除基质组织，导致角膜样本更薄，从而减少基质肿胀（图1和图2）。较少基质肿胀诱导较少的剪切和捏力，对角膜内皮有负面影响，从而导致较低的内皮细胞损失率6。与非分裂的角膜纽扣和整个角膜样本相比，分离角膜按钮在15天的培养后明显具有更好的保存性内皮细胞层，这反?…
Discussion
该协议提供了一种制备猪分裂角膜纽扣的方法，它代表了一种标准化和低成本的外体角膜内皮器官培养模型，用于研究目的6。猪分裂角膜纽扣显示内皮细胞密度下降，与在眼库培养的人类供体角膜中观察到的内皮细胞损失相当，在两周内6，10，11， 12.
优于非分?…
Declarações
The authors have nothing to disclose.
Acknowledgements
所提供的研究模型的建立得到了德国联邦教育和研究部的KMU-创新（FKZ：13GW0037F）的支持。
Materials
| Subject | |||
| Pig eyes | local abbatoir | ||
| Substances | |||
| Alizarin red S | Sigma-Aldrich, USA | ||
| Culture Medium 1, #F9016 | Biochrom GmbH, Germany | ||
| Dulbecco's PBS (1x) | Gibco, USA | ||
| Fetal calf serum | Biochrom GmbH, Germany | ||
| Hydrochloric acid (HCl) solution | own production | ||
| Hypotonic balanced salt solution | own production | per 1 L of H2O: NaCl 4.9 g; KCl 0.75 g; CaCl x H2O 0.49 g; MgCl2 x H2O 0.3 g; Sodium Acetate x 3 H2O 3.9 g; Sodium Citrate x 2 H2O 1.7 g | |
| Povidon iodine 7.5%, Braunol | B. Braun Melsungen AG, Germany | ||
| Sodium chloride (NaCl) 0.9% | B. Braun Melsungen AG, Germany | ||
| Sodium hydroxide (NaOH) solution | own production | ||
| Trypan blue 0.4% | Sigma-Aldrich, USA | ||
| Materials & Instruments | |||
| Accu-jet pro | Brand GmbH, Germany | ||
| Beaker Glass 50 mL | Schott AG, Germany | ||
| Blunt cannula incl. Filter (5 µm) 18G | Becton Dickinson, USA | ||
| Cell culture plate (12 well) | Corning Inc., USA | ||
| Colibri forceps | Geuder AG, Germany | ||
| Corneal scissors | Geuder AG, Germany | ||
| Eppendorf pipette | Eppendorf AG, Germany | ||
| Eye Bulb Holder | L. Klein, Germany | ||
| Eye scissors | Geuder AG, Germany | ||
| Folded Filter ø 185 mm | Whatman, USA | ||
| Hockey knife | Geuder AG, Germany | ||
| Laboratory Glass Bottle with cap 100 mL | Schott AG, Germany | ||
| Magnetic stir bar | Carl Roth GmbH & Co. KG, Germany | ||
| MillexGV Filter (5 µm) | Merck Millopore Ltd., USA | ||
| Needler holder | Geuder AG, Germany | ||
| Petri dishes | VWR International, USA | ||
| Pipette tips | Sarstedt AG & Co., Germany | ||
| Scalpel (single use), triangular blade | Aesculap AG & Co. KG, Germany | ||
| Serological pipette 10 mL | Sarstedt AG & Co., Germany | ||
| Serological pipette 5 mL | Sarstedt AG & Co., Germany | ||
| Sterile cups | Greiner Bio-One, Österreich | ||
| Sterile gloves | Paul Hartmann AG, Germany | ||
| Sterile surgical drape | Paul Hartmann AG, Germany | ||
| Stitch scissors | Geuder AG, Germany | ||
| Suture Ethilon 10-0 Polyamid 6 | Ethicon Inc., USA | ||
| Syringe (5 mL) | Becton Dickinson, USA | ||
| trephine ø 7.5 mm | own production | ||
| Tying forceps | Geuder AG, Germany | ||
| Weighing paper | neoLab Migge GmbH, Germany | ||
| Equipment & Software | |||
| Binocular surgical microscope | Carl Zeiss AG, Germany | ||
| Camera mounted on microscope | Olympus, Japan | ||
| CellSens Entry (software) | Olympus, Japan | ||
| Cold-light source | Schott AG, Germany | ||
| Incubator | Heraeus GmbH, Germany | ||
| Inverted phase contrast microscope | Olympus GmbH, Germany | ||
| Magnetic stirrer with heating function | IKA-Werke GmbH & Co. KG, Germany | ||
| pH-meter pHenomenal | VWR International, USA | ||
| Photoshop CS2 | Adobe Systems, USA | ||
| Precision scale | Ohaus Europe GmbH, Switzerland |
Referências
- Gain, P., et al. Global Survey of Corneal Transplantation and Eye Banking. JAMA Ophthalmology. 134 (2), 167-173 (2016).
- Roy, O., et al. Understanding the process of corneal endothelial morphological change in vitro. Investigative Ophthalmology & Visual Science. 56 (2), 1228-1237 (2015).
- Meltendorf, C., Ohrloff, C., Rieck, P., Schroeter, J. Endothelial cell density in porcine corneas after exposure to hypotonic solutions. Graefe’s Archive for Clinical and Experimental Ophthalmology. 245 (1), 143-147 (2007).
- Schroeter, J., Meltendorf, C., Ohrloff, C., Rieck, P. Influence of temporary hypothermia on corneal endothelial cell density during organ culture preservation. Graefe’s Archive for Clinical and Experimental Ophthalmology. 246 (3), 369-372 (2008).
- Schroeter, J., Ruggeri, A., Thieme, H. Impact of temporary hyperthermia on corneal endothelial cell survival during organ culture preservation. Graefe’s Archive for Clinical and Experimental Ophthalmology. 253 (5), 753-758 (2015).
- Kunzmann, B. C., et al. Establishment Of A Porcine Corneal Endothelial Organ Culture Model For Research Purposes. Cell and Tissue Banking. 19 (3), 269-276 (2018).
- Redbrake, C., et al. A histochemical study of the distribution of dextran 500 in human corneas during organ culture. Current Eye Research. 16 (5), 405-411 (1997).
- Zhao, M., et al. Poloxamines for Deswelling of Organ-Cultured Corneas. Ophthalmic Research. 48 (2), 124-133 (2012).
- Filev, F., et al. Semi-quantitative assessments of dextran toxicity on corneal endothelium: conceptual design of a predictive algorithm. Cell and Tissue Banking. 18 (1), 91-98 (2017).
- Pels, E., Schuchard, Y. Organ-culture preservation of human corneas. Documenta Ophthalmologica. 56 (1-2), 147-153 (1983).
- Borderie, V. M., Kantelip, B. M., Delbosc, B. Y., Oppermann, M. T., Laroche, L. Morphology, Histology and Ultrastructure of Human C31 Organ-Cultured Corneas. Cornea. 14 (3), 300-310 (1995).
- Linke, S. J., et al. Thirty years of cornea cultivation: long-term experience in a single eye bank. Acta Opthalmologica. 91 (6), 571-578 (2013).
- Schroeter, J., et al. Arbeitsrichtlinien – Gute Fachliche Praxis für Hornhautbanken [Procedural guidelines. Good tissue practice for cornea banks]. Ophthalmologe. 106 (3), 265-276 (2009).
- Dohlman, C. H., Hedbys, B. O., Mishima, S. The swelling pressure of the corneal stroma. Investigative Ophthalmology & Visual Science. 1, 158-162 (1962).
- Xuan, M., et al. Proteins of the corneal stroma: importance in visual function. Cell and Tissue Research. 364 (1), 9-16 (2016).
- Sperling, S. Human Corneal Endothelium in Organ Culture – The Influence of Temperature and Medium of Incubation. Acta Opthalmologica. 57 (2), 269-276 (1979).
- Schroeter, J. Endothelial Evaluation in the Cornea Bank. Developments in Ophthalmology. 43, 47-62 (2009).
- Pels, E., Schuchard, Y. The Effects of High Molecular Weight dextran on the Presevation of Human Corneas. Cornea. 3 (3), 219-227 (1985).
- van der Want, H. J. L., Pels, E., Schuchard, Y., Olesen, B., Sperling, S. Electron Microscopy of Cultured Human Corneas Osmotic Hydration and the Use of dextran Fraction (dextran T 500) in Organ Culture. Archives of Ophthalmology. 101 (12), 1920-1926 (1983).
- Thuret, G., Manissolle, C., Campos-Guyotat, L., Guyotat, D., Gain, P. Animal compound-free medium and poloxamer for human corneal organ culture and Deswelling. Investigative Ophthalmology & Visual Science. 46 (3), 816-822 (2005).
- Wenzel, D. A., Kunzmann, B. C., Spitzer, M. S., Schultheiss, M. Staining of endothelial cells does not change the result of cell density. Cell and Tissue Banking. 20 (2), 327-328 (2019).
- Wenzel, D. A., Kunzmann, B. C., Hellwinkel, O., Druchkiv, V., Spitzer, M. S., Schultheiss, M. Effects of perfluorobutylpentane (F4H5) on corneal endothelial cells. Current Eye Research. , (2019).
- Olsson, I. A. S., Franco, N. H., Weary, D. M., Sandøe, P. The 3Rs principle – mind the ethical gap!. ALTEX Proceedings, 1/12, Proceedings of WC8. , 333-336 (2012).
- Sanchez, I., Martin, R., Ussa, F., Fernandez-Bueno, I. The parameters of the porcine eyeball. Graefe’s Archive for Clinical and Experimental Ophthalmology. 249 (4), 475-482 (2011).
- Kim, M. K., Hara, H. Current status of corneal xenotransplantation. International Journal of Surgery. 23 (Pt B), 255-260 (2015).
- Fujita, M., et al. Comparison of Proliferative Capacity of Genetically-Engineered Pig and Human. Ophthalmic Research. 49 (3), 127-138 (2013).
Citar este artigo
Wenzel, D. A., Kunzmann, B. C., Steinhorst, N. A., Spitzer, M. S., Schultheiss, M. A Porcine Corneal Endothelial Organ Culture Model Using Split Corneal Buttons. J. Vis. Exp. (152), e60171, doi:10.3791/60171 (2019).